The fluorescence line shape of the intrinsic fluorophore of the green fluorescent protein depends not only on its chemical identity, but also on its environment. The aim of the proposed research is to investigate the relationships between this line shape and the conformational flexibility of this environment by entering a cycle of high throughput spectroscopy, protein sequencing and computation. Computation will first be used to design a cell-based library enriched with functional fluorescent proteins. Flow cytometry will then be used to screen more than 10/8 members of this library for proteins with exceptional fluorescence line shapes. Hundreds of mutants will then be sequenced following a second screen using the increased spectral resolution of an automated fluorimeter. The cycle will be completed by estimating the entropies of the different sequences and by investigating the relationships between the values calculated and the spectra observed. Both the improved understanding of spectroscopic structure-function relationships and the development of novel screens will facilitate the ongoing search for fluorophores optimized for biological research and medical diagnostics applications. Insights gained may be transferable to engineering other functions of proteins that depend on their electronic structure and dynamics, such as enzymatic catalysis.